JP2001178471A - Method for immobilizing dna fragment to surface of solid- phase carrier and dna chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To develop an immobilization method capable of bonding a DNA fragment separately prepared beforehand to the surface of a solid-phase carrier by a rapid reaction and making the reaction product stably maintain the bond and to obtain a DNA chip not requiring a blocking process. SOLUTION: This method for immobilizing the DNA fragment to the surface of solid-phase carrier is characterized in that a DNA fragment containing an active methylene group at a terminal end is brought into contact in a liquid phase with a solid-phase carrier having the surface to which a diazonium base- containing chain molecule is fixed at one terminal end so that a bond occurs between the DNA fragment and the chain molecule. This DNA chip is obtained by the method. This method for detecting a nucleic acid fragment having complementarity to the DNA fragment on the DNA chip uses the DNA chip.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a large number of DNs which are very useful for simultaneous analysis of gene expression, mutation, polymorphism, etc.
The present invention relates to a method for immobilizing DNA fragments on a solid-phase carrier surface, which is necessary for producing a high-density array (DNA chip) in which A fragments and oligonucleotides are arranged on a solid-phase surface. The present invention also provides a DNA chip produced by a method for immobilizing the DNA fragment on the surface of a solid support, and DN on the DNA chip.
The present invention also relates to a method for detecting a nucleic acid fragment having complementarity to the A fragment.

[0002]

2. Description of the Related Art Technology development for efficiently analyzing the functions of all genes of various organisms has been progressing, and a DNA chip has been used as an analysis means. A DNA chip is usually in the form of a microarray in which a large number of DNA fragments are aligned and fixed on a solid support such as a slide glass, and has a DNA complementary to the DNA fragments fixed to the DNA chip.
The fragment sample is immobilized on a DNA chip by hybridization and used for detection. Known methods for detecting the formed hybrid include a method using a fluorescent label or a radioactive label previously bound to a DNA fragment sample, and a method using an intercalator having a conductive group incorporated into the hybrid. .

[0003] DNA chip technology using a DNA chip can be applied to biomolecules other than DNA, and provides new means for research and development of drug discovery research, diagnosis and prevention of diseases, and measures against energy and environmental problems. It is expected to provide.

[0004] The use of a DNA chip as a DNA analysis means has been embodied in a method of determining the base sequence of DNA by hybridization with an oligonucleotide (SBH, sequencing by hyb).
(D. Ridation) is devised (D
rmanac, R .; et al. , Genomics,
4, page 114 (1989)). Although SBH was a method capable of overcoming the limitations of base sequencing using gel electrophoresis, it did not reach practical use.

[0005] Subsequently, a DNA chip fabrication technique was developed, and so-called HTS (high throughppu), which efficiently and efficiently examines gene expression, mutation, polymorphism and the like in a short time.
t screening) (Fodo
r, S. P. A. , Science, 251, page
767 (1991) and Schena, M .; , Sc
issue, 270, page 467 (199
5)).

However, in order to put the DNA chip utilization technology into practical use, a DNA chip production technology for aligning and immobilizing a large number of DNA fragments and oligonucleotides on the surface of a solid support is required.

As a method for preparing a DNA chip, a method of directly synthesizing a DNA fragment on the surface of a solid support (referred to as an “on-chip method”) and a method of fixing a separately prepared DNA fragment on the surface of a solid support are used. And is known. The on-chip method combines the use of protecting groups that are selectively removed by light irradiation with the photolithography and solid-phase synthesis techniques used in semiconductor manufacturing to define a specific area of a tiny matrix. A method of performing selective synthesis (referred to as “masking technique”) is typical.

As a method for immobilizing a DNA fragment prepared in advance on the surface of a solid support, there are the following methods depending on the type of the DNA fragment and the type of the solid support. (1) The DNA fragment to be fixed is cDNA (complementary DNA synthesized using mRNA as a template) or PCR product (cDNA
In the case of a DNA fragment obtained by amplifying the DNA fragment by a PCR method, these are applied to the surface of a solid support that has been surface-treated with a polycation (polylysine, polyethyleneimine, etc.) using a spotter device provided in a DNA chip preparation device. In general, a method of spotting and electrostatically bonding to a solid support using the charge of DNA is used. As a method for treating the surface of the solid support, a method using a silane coupling agent having an amino group, an aldehyde group, an epoxy group, or the like is also used (Geo, Z. et al., Nucleic A).
cid Research, 22, 5456-5465
(1994)). In this case, since amino groups, aldehyde groups, and the like are introduced into the surface of the solid support by covalent bonds, they are more stably present on the surface of the solid support than in the case of polycations.

As a modification of the method utilizing the charge of DNA, a PCR product modified with an amino group is suspended in SSC (standard saline citrate buffer) and spotted on a silylated slide glass surface. A method of sequentially performing a treatment with sodium borohydride and a heat treatment after incubation (Schena, M. et a) has been reported.
l. Proc. Natl. Acad. Sci. USA
93, 10614-10619 (1996)). However, this fixing method has a problem that sufficient stability is not always obtained. In DNA chip technology, the detection limit is important. Therefore, the development of a technique for stably immobilizing a DNA fragment in a sufficient amount on the surface of a solid support greatly contributes to improving the detection limit of hybridization between the immobilized DNA fragment and the labeled sample nucleic acid fragment.

(2) When the DNA fragment to be fixed is a synthetic oligonucleotide, an oligonucleotide having a reactive group introduced therein is synthesized, the oligonucleotide is spotted on the surface of the surface-treated solid support, and covalently bonded. ("Protein / Nucleic acid / Enzyme", Vol. 43, (1998), 2004-2)
011, Lamture, J.M. B. et al. , Nu
cl. Acids Res. , 22,2121-212
5, 1994, and Guo. Z. , Et al. ,
Nucl. Acids Res. , 22,5456-5
465, 1994). For example, a method of reacting an amino group-introduced oligonucleotide with a slide glass having an amino group introduced thereto in the presence of PDC (p-phenylenediisothiocyanate) and a method of reacting an aldehyde group-introduced oligonucleotide with the slide glass are known. Have been. In these two methods, the oligonucleotide is stably immobilized on the surface of the solid-phase carrier as compared with the method (1) using the charge of DNA. However, in the method in which PDC is present, the reaction between the PDC and the amino group-introduced oligonucleotide is slow, and in the method using the aldehyde group-introduced oligonucleotide, the stability of the reaction product, the Schiff base, is low (usually, hydrolysis). Is more likely to occur). Furthermore, DNA such as amino groups
If a functional group having a strong interaction with the nucleic acid exists on the entire surface, the nucleic acid fragment as a test substance is likely to non-specifically adhere to the entire surface of the DNA chip, and thus there is a problem that detection is hindered. For this reason, in order to prevent this, a step called blocking, which blocks unreacted functional groups, was required.

[0011]

DISCLOSURE OF THE INVENTION The present invention relates to a method for binding a separately prepared DNA fragment to the surface of a solid support by a rapid reaction, and to stably maintain the binding of the reaction product. It is an object of the present invention to provide an immobilization method, a DNA chip which does not particularly require a blocking step, and a method for detecting a nucleic acid fragment.

[0012]

The above objects have been attained by the present invention described below.

(1) D having an active methylene group at the terminal
The NA fragment is brought into contact with a solid phase carrier having a chain molecule having a diazonium base fixed at one end to the surface in a liquid phase to form a bond between the DNA fragment and the chain molecule. A method for immobilizing a DNA fragment on a surface of a solid support. In the fixing method of the present invention, the contact is preferably performed in the presence of an aqueous liquid having a pH of 4 to 11, and the contact is preferably performed in the presence of an aqueous liquid having a pH of 6 to 10. It is particularly preferable to carry out the above. (2) A DNA chip obtained by the above method.

(3) A step of applying an aqueous liquid containing a nucleic acid fragment sample labeled with a fluorescent substance or a radioactive substance to the surface of the DNA chip, a nucleic acid fragment complementary to the DNA fragment fixed to the DNA chip Immobilizing the sample on a DNA chip by hybridization,
And detecting a fluorescent label or a radioactive label of the labeled nucleic acid fragment sample immobilized on the DNA chip.
A method for detecting a nucleic acid fragment having complementarity to a DNA fragment on a DNA chip. (4) a step of applying an aqueous liquid containing an intercalator having a conductive group and a nucleic acid fragment sample to the surface of the DNA chip, and removing the nucleic acid fragment sample complementary to the DNA fragment fixed to the DNA chip; Immobilizing on a DNA chip by hybridization, and DNA
A step of electrochemically detecting a current flowing through a conductive group of an intercalator incorporated into a hybrid structure formed from a DNA fragment and a nucleic acid fragment sample of the chip, For detecting nucleic acid fragments having complementary nature.

Preferred embodiments of the method for immobilizing a DNA fragment on the surface of a solid support of the present invention are as follows. (1) A diazonium salt is immobilized on a solid support. (2) the nucleotide sequence is known as a DNA fragment;
One having a partial structure having an active methylene group at one end thereof is reacted with the above-mentioned solid support for immobilizing a DNA fragment carrying the above diazonium salt.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The solid support for immobilizing a DNA fragment of the present invention comprises a solid support and a diazonium salt compound supported on the solid support.
The NA fragment is immobilized to obtain a DNA-immobilized solid support.

FIG. 1 of the accompanying drawings schematically shows an embodiment. In order to produce the DNA fragment-immobilized solid phase carrier (hereinafter referred to as “DNA chip”) of the present invention, first, a reactive group is introduced into the solid phase carrier (M) via a linking group (L). The carrier (1) is prepared, and the diazonium salt compound is allowed to act on the carrier (1) to guide the solid carrier (1) to the solid carrier (2). Then, by bringing a DNA fragment having an active methylene group into contact with the solid support (2), D
An NA chip (3) can be obtained.

The structural characteristics of the compound capable of diazo coupling with a diazonium salt are as described below, and compounds belonging to the group of compounds used as so-called color photographic couplers are preferred. Examples of photographic couplers include, for example, 1977 Macmillan Publishing Co., Inc. (New York)
Published by THJames, The Theory of the Photographic
Process, 4th eddition, Chapter 12, Section 3, pages 352-362, in a review by JRThirtle.

The diazonium salt compound is preferably directly linked to the solid support by a covalent bond from the viewpoint of simplicity of the linking operation. However, the diazonium salt compound is linked to the solid support by a covalent bond to a compound previously covalently linked to the solid support. You may. Alternatively, it is also possible to carry the compound by utilizing electrostatic interaction with a polyanionic polymer previously carried on the surface of the solid support. Alternatively, a diazonium salt compound can be obtained by immobilizing a precursor of a compound that generates a diazonium salt such as an aromatic amine on the surface of a solid support and then subjecting the precursor to a diazotization reaction treatment. The diazotization reaction of an aromatic amine can be carried out according to a conventional method of diazotization, for example, by reacting nitrite under acidic conditions or by reacting a nitrite such as isoamyl nitrite.

In order to link the diazonium salt compound or its precursor to the surface of the solid support through a covalent bond, a known synthesis reaction can be used. Examples of the reaction include a reaction between a glass surface and a silane coupling agent, a reaction between a functional group on a synthetic resin surface and an active halide, and a reaction between a cellulose resin and an isocyanate.

Examples of the reaction with a reactive group previously introduced on the surface of the solid support include a nucleophilic group (amino group, hydroxy group, etc.) and an electron accepting group (carboxyl group, ester group, isocyanate group, A combination with an isothiocyanate group, a conjugated ketone, an acrylate ester, vinyl sulfone, an acid anhydride, an acyl halide, a sulfonyl halide, and the like.

Hereinafter, specific examples of the diazonium salt compound or its precursor on the surface of the solid support in the present invention will be described.
The scope of the present invention is not limited only by these. Here, M represents a solid support. The sum of m and n is
100 mol%.

[0023]

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[0024]

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In the following, specific examples of a partial structure containing an active methylene group to be incorporated into a DNA fragment for coupling with a diazonium salt in the present invention are shown, but the scope of the present invention is not limited only to these. Absent. The bond shown below indicates a position at which the bond to the DNA fragment.

[0026]

Embedded image

As the solid phase carrier, a carrier having low hydrophobicity or low hydrophilicity is preferable. In addition, even if the surface has unevenness and low flatness, it can be preferably used. Materials for the solid support include ceramics or new ceramics such as glass, cement, and porcelain, polymers such as polyethylene terephthalate, cellulose acetate, bisphenol A polycarbonate, polystyrene, and polymethyl methacrylate, silicon, activated carbon, porous glass, and porous materials. Ceramics, porous silicon, porous activated carbon, woven fabric, knitted fabric, nonwoven fabric, filter paper, short fibers, porous materials such as membrane filters, conductive materials such as gold, etc. can be used. The size of the pores of the porous material is preferably in the range of 2 to 1000 nm, particularly preferably in the range of 2 to 500 nm. The material of the solid support is particularly preferably glass or silicon. This is due to the ease of surface treatment and the ease of analysis by electrochemical methods. The thickness of the solid support is preferably in the range of 100 to 2000 μm.

The surface of the solid support may be coated with a polymer such as a polycation or the like, or may be treated with a silane coupling agent having a substituent introduced to the surface of the solid support. Alternatively, a reactive functional group may be introduced to the surface by plasma treatment. In the case of polycations, anionic species can be imperfectly retained on the surface of the solid support by electrostatic bonding, but in the case of silane coupling agents, various compounds can be covalently bonded to the surface of the solid support. Is preferred because it can be stably bonded to Thus, an amino group, a carboxyl group, a hydroxy group, or the like can be introduced. A compound having a photoreactive group may be linked to these introduced groups via a covalent bond. Examples of silane coupling agents include γ-
Preferably, aminopropyltriethoxysilane, N-β- (aminoethyl) -γ-aminopropyltrimethoxysilane or N-β- (aminoethyl) -β-aminopropylmethyldimethoxysilane is used. It is particularly preferred to use ethoxysilane.

The treatment using a polycation may be combined with the treatment using a silane coupling agent. It is possible to promote electrostatic interaction between the DNA fragment and the solid support having low hydrophobicity or low hydrophilicity. A layer made of a charged hydrophilic polymer or the like or a layer made of a cross-linking agent may be further provided on the surface-treated solid support surface.
By providing such grooves, it is possible to reduce unevenness of the solid support having been subjected to the surface treatment. Depending on the type of the solid phase carrier, a hydrophilic polymer or the like can be contained in the carrier, and a solid phase carrier that has been subjected to such treatment can be preferably used.

The DNA fragment can be divided into two types depending on the purpose. To examine gene expression, the cD
It is preferable to use a polynucleotide such as NA, a part of cDNA, or EST. These polynucleotides may be of unknown function, but are generally amplified by PCR using a cDNA library, genomic library or whole genome as a template based on sequences registered in a database. (Hereinafter, referred to as “PCR product”). Those not amplified by the PCR method can also be preferably used. In addition, in order to examine gene mutations and polymorphisms, it is preferable to synthesize various oligonucleotides corresponding to the mutations and polymorphisms based on a known sequence as a standard, and use them. Furthermore, in the case of nucleotide sequence analysis, it is preferable to use 4 ⁿ (n is the length of a base) synthesized oligonucleotides. The base sequence of the DNA fragment is preferably determined in advance by a general base sequence determination method. The DNA fragment is preferably a 2- to 50-mer, and particularly preferably a 10- to 25-mer.

An active methylene group for forming a bond by diazo coupling with a diazonium salt on the surface of a solid support is introduced into one end of the DNA fragment directly or via a linking group. Examples of these are as described above.

The DNA fragment can be spotted by dissolving or dispersing the DNA fragment in an aqueous medium in a 96-well or 3-well solution.
The dispensing is preferably performed by dispensing into an 84-well plastic plate and dropping the dispensed aqueous liquid onto the surface of the solid support using a spotter or the like.

In order to prevent drying of the DNA fragment after spotting,
A substance having a high boiling point may be added to the aqueous liquid in which the DNA fragment is dissolved or dispersed. As a substance with a high boiling point,
It is preferably a substance which can be dissolved in an aqueous liquid in which a DNA fragment is dissolved or dispersed, and which does not hinder hybridization with a sample nucleic acid fragment and has low viscosity. Such materials can include glycerin, ethylene glycol, dimethyl sulfoxide and low molecular weight hydrophilic polymers. Examples of the hydrophilic polymer include polyacrylamide, polyethylene glycol, and sodium polyacrylate. The molecular weight of the polymer is preferably in the range of 10 ³ to 10 ^6. As the substance having a high boiling point, glycerin or ethylene glycol is more preferably used, and glycerin is particularly preferably used.
The concentration of the high-boiling substance is 0.1% in the aqueous solution of the DNA fragment.
Preferably, it is in the range of 0.5 to 2% by volume, particularly preferably in the range of 0.5 to 1% by volume. Further, for the same purpose, the solid support after spotting the DNA fragment,
It is also preferable to place in an environment with a humidity of 90% or more and a temperature range of 25 to 50 ° C.

After spotting the DNA fragment, diazo coupling with a diazonium salt is performed. At this time, the pH of the spotting solution is preferably set to be equal to or higher than the acid dissociation constant of the coupler, for the purpose of promoting the diazo coupling by assisting the coupler to dissociate protons to anions to promote anion. More preferably, the pH is set within the range.
It is particularly preferable to set it in the range of 10 to 10. Then, by washing away the non-immobilized DNA,
The DNA fragment can be immobilized on the surface of the solid support in any shape.

After spotting the DNA fragment, in addition to performing the diazo coupling reaction, post-treatment with sodium borohydride or a Schiff reagent may be performed. These post-treatments may be performed in combination of a plurality of types, and particularly preferably performed in combination of the heat treatment and the ultraviolet treatment. These post-treatments are particularly effective when the surface of the solid support is treated only with the polycation. After spotting, it is also preferable to carry out incubation. After the incubation, it is preferable to wash and remove unspotted DNA fragments.

The DNA fragment is applied to the surface of the solid support at 1
It is preferably in the range of 0 ^{2 to} 10 ⁵ types / cm ² . The amount of the DNA fragment is in the range of 1 to 10 ¹⁵ mol, and the weight is preferably several ng or less. By spotting, the aqueous liquid of the DNA fragment is fixed in the form of dots on the surface of the solid support. The shape of the dot is almost circular. The absence of variation in shape is important for quantitative analysis of gene expression and analysis of single nucleotide mutation. The distance between the dots is preferably in the range of 0 to 1.5 mm, particularly preferably in the range of 100 to 300 μm. The size of one dot is 50 to 3 in diameter.
It is preferably in the range of 00 μm. The amount of spotting is
It is preferably in the range of 100 pL to 1 μL,
It is particularly preferred that it is in the range of 100 to 100 nL.

The life of the DNA chip prepared by the above-described process is as follows.
Longer for chips. These DNA chips are used for gene expression monitoring, nucleotide sequence determination, mutation analysis, polymorphism analysis, and the like. The principle of detection is hybridization with a labeled sample nucleic acid fragment described below.

As the weaving method, an RI method and a non-RI method (fluorescence method, biotin method, electrochemical method, chemiluminescence method, etc.) are known, but the fluorescence method is used in the present invention. As the fluorescent substance, any substance can be used as long as it can bind to the base portion of the nucleic acid.
CyDye ^™ series Cy3, Cy5, etc.), rhodamine 6G reagent, N-acetoxy-N ² -acetylaminofluorene (AAF) or AAIF (iodine derivative of AAF) can be used.

It is to be noted that, besides utilizing the above-mentioned label, a method utilizing an electrochemical detection method using an intercalator having a conductive group and having a property of being incorporated into the formed hybrid structure is also known. And D of the present invention
NA chips can also be used for electrochemical detection methods. Alternatively, there is also known a method of using a method for detecting the formation of a hybrid by a fluorescence method using an intercalator having a fluorescence-generating group and having a property of being incorporated into the formed hybrid structure. Can also be used for this detection method.

As the sample nucleic acid fragment, it is preferable to use a DNA fragment sample or an RNA fragment sample whose sequence or function is unknown. The sample nucleic acid fragment is preferably isolated from eukaryotic cell or tissue samples for the purpose of examining gene expression. If the sample is a genome, it is preferably isolated from any tissue sample except red blood cells. Any tissue other than red blood cells is preferably peripheral blood lymphocytes, skin, hair, semen and the like. If the sample is mRNA, it is preferably extracted from a tissue sample in which mRNA is expressed. mRNA is labeled dN by reverse transcription reaction.
It is preferable that TP ("dNTP" means a deoxyribonucleotide whose base is adenine (A), cytosine (C), guanine (G) or thymine (T)) is incorporated into a labeled cDNA. As dNTP, it is preferable to use dCTP for chemical stability. MRN required for one hybridization
The amount of A varies depending on the amount of the liquid and the labeling method, but is preferably several μg or less. When the DNA fragment on the DNA chip is an oligo DNA, it is desirable that the sample nucleic acid fragment be reduced in molecular weight. In prokaryotic cells, m
Since it is difficult to selectively extract RNA, it is preferable to label total RNA. The sample nucleic acid fragment may be labeled with a labeled primer or labeled dN for the purpose of examining gene mutation or polymorphism.
It is preferable to obtain by carrying out PCR of the target region in a reaction system containing TP.

In the hybridization, an aqueous solution, in which a labeled sample nucleic acid fragment is dissolved or dispersed, dispensed on a 96-well or 384-well plastic plate is spotted on the DNA chip prepared above. It is preferable to carry out the method. The amount of spotting is 1 to 10
It is preferably in the range of 0 nL. Hybridization is preferably carried out in a temperature range from room temperature to 70 ° C. and for a time in the range from 6 to 20 hours. After the completion of the hybridization, it is preferable to wash with a mixed solution of a surfactant and a buffer to remove unreacted sample nucleic acid fragments. It is preferable to use sodium dodecyl sulfate (SDS) as the surfactant. As a buffer, a citrate buffer, a phosphate buffer, a borate buffer, a Tris buffer, a Good buffer, and the like can be used, but a citrate buffer is particularly preferable.

A feature of hybridization using a DNA chip is that the amount of labeled sample nucleic acid fragments used is very small. Therefore, DN immobilized on a solid support
It is necessary to set optimal hybridization conditions depending on the length of the A fragment and the type of the labeled sample nucleic acid fragment. For gene expression analysis, it is preferable to perform hybridization for a long time so that low-expressed genes can be sufficiently detected. For detection of a single nucleotide mutation, it is preferable to perform hybridization for a short time. Another feature is that by preparing two types of sample nucleic acid fragments labeled with different fluorescent substances and simultaneously using them for hybridization, the expression level can be compared and quantified on the same DNA chip.

[0043]

EXAMPLES Example 1 Preparation of DNA Fragment-Immobilized Slide and Measurement of Amount of Immobilized DNA Fragment The immobilization method of the present invention is represented by a DNA fragment reaction path, and the reaction path is shown in FIG. In FIG. 2, M represents a slide glass.

(1) Preparation of a slide (B) into which a diazonium salt was introduced A slide glass (25) was added to an ethanol solution of 2 mass% aminopropylethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd.).
(mm × 75 mm) was taken out after dipping for 10 minutes, washed with ethanol, and dried at 110 ° C. for 10 minutes to prepare a silane compound-coated slide (A). Then, the slide (A) coated with the silane compound was placed in the presence of a condensing agent,
N of benzyloxycarbonylaminobenzoic acid (1 g)
After being immersed in a methylpyrrolidone (50 mL) solution for 1 hour, it was taken out and washed with acetonitrile. Then, it was immersed in a 1M aqueous hydrogen chloride solution (50 mL) at 50 ° C. for 1 hour, and then immersed in an ice-cooled 1M hydrogen chloride aqueous solution (50 mL).
After adding 0.5 g of sodium nitrite and leaving the mixture to stand for 10 minutes, it was taken out, the liquid was absorbed by filter paper, and a slide (B) into which diazonium salt was introduced was obtained.

(2) Spotting of DNA Fragments and Measurement of Fluorescence Intensity The 3 ′ end and 5 ′ end were each an acetoacetylamino group, and a fluorescent labeling reagent (FluoroLink Cy5dC) was used.
TP, manufactured by Amersham Pharmacia Biotech)
DNA fragment modified with (3'CTAGTCTGTGAAGTGTCTGATC
An aqueous liquid (1 × 10 ⁻⁶ M, 1 μL) obtained by dispersing 5 ′) in a 0.1 M carbonate buffer (pH 9.3) is spotted on the slide (B) obtained in the above (1). Slide 0.1
Mass% SDS (sodium dodecyl sulfate) and 2 × SSC
(2 × SSC: a solution obtained by diluting the stock solution of SSC by 2 times, S
SC: standard saline citrate buffer) twice.
Washing was sequentially performed once with a 0.2 × SSC aqueous solution. Next, the slide after the above washing was washed with a 0.1 M glycine aqueous solution (pH
10) After immersion for 1 hour and 30 minutes, washed with distilled water,
After drying at room temperature, a slide (C
1) was obtained. The fluorescence intensity on the surface of the slide (C1) was measured by a fluorescence scanning apparatus and was found to be 1893. This value is extremely large as compared with the following comparative examples, and it is understood that the DNA fragment was efficiently fixed to the slide glass by the immobilization method of the present invention.

Comparative Example 1 A slide (C2) was prepared in the same manner as in Example 1 except that sodium nitrite was not used. The fluorescence intensity of the surface of the slide (C2) was measured by a fluorescence scanning apparatus and was found to be 204. That is, it can be seen that the amount of immobilized DNA fragments is much smaller than in the examples.

Example 2 Detection of Sample DNA Fragment (1) Preparation of DNA Chip A DNA fragment was immobilized in the same manner as in Example 1 except that a DNA fragment whose 3 ′ end was not modified with a fluorescent labeling reagent was used. A slide (D2) was obtained. (2) Detection of Sample DNA Fragment A 22-mer sample oligonucleotide (GATCAGACACTTCACAGACTAG5 ′) with Cy5 bound to the 5 ′ end was dispersed in a hybridization solution (a mixed solution of 4 × SSC and 10% by weight SDS) (20 μL). What
The sample was spotted on the slide (D2) obtained in (1) above, the surface was protected with a microscope cover glass, and the plate was incubated at 60 ° C. for 20 hours in a Moisture chamber. Next, this was sequentially washed with a mixed solution of 0.1% by mass SDS and 2 × SSC, a mixed solution of 0.1% by mass SDS and 0.2 × SSC, and a 0.2 × SSC aqueous solution. 6
Centrifuged at 00 rpm for 20 seconds and dried at room temperature. The fluorescence intensity on the surface of the slide glass was measured by a fluorescence scanning device and was found to be 1629. By using the DNA chip prepared by the immobilization method of the present invention,
It can be seen that a sample DNA fragment having complementarity with the DNA fragment immobilized on the DNA chip can be detected.

[0048]

According to the present invention, DNA can be deposited on the surface of a solid support.
The fragments can be fixed stably and quickly. In particular, when a diazonium salt compound is introduced into the surface of a solid support using a silane coupling agent, both the binding of the diazonium salt compound to the surface of the solid support and the diazo bond with the DNA fragment are covalent bonds. Therefore, the DNA fragment can be firmly fixed. The stable immobilization of DNA fragments enables the production of a DNA chip having a high detection limit that can be effectively used for gene analysis and the like. As one example, by performing hybridization with a sample nucleic acid fragment using a DNA chip prepared according to the present invention, a sample nucleic acid fragment having complementarity to a DNA fragment immobilized on a DNA chip can be detected with high sensitivity. Can be detected.

[Brief description of the drawings]

FIG. 1 is a schematic view showing a typical fixing method of the present invention.

FIG. 2 is a schematic diagram showing the method for immobilizing a DNA fragment of the present invention.

[Explanation of symbols]

 M slide glass

Claims (6)

[Claims] 1. A DNA having an active methylene group at the terminal
Forming a bond between the DNA fragment and the chain molecule by contacting the fragment and a solid carrier having a diazonium base-containing chain molecule fixed to the surface at one end in a liquid phase; A method for immobilizing a DNA fragment on a surface of a solid support, which is characterized in that: 2. The method according to claim 1, wherein the contacting is performed in the presence of an aqueous liquid having a pH in the range of 4 to 11. 3. The method according to claim 2, wherein the contacting is performed in the presence of an aqueous liquid having a pH in the range of 6 to 10. 4. A DNA chip obtained by the method according to claim 1. 5. A step of applying an aqueous liquid containing a nucleic acid fragment sample labeled with a fluorescent substance or a radioactive substance to the surface of the DNA chip according to claim 4, wherein complementarity with the DNA fragment immobilized on the DNA chip is determined. Immobilizing a nucleic acid fragment sample having on a DNA chip by hybridization, and detecting a fluorescent label or a radioactive label of the labeled nucleic acid fragment sample immobilized on the DNA chip. A method for detecting a nucleic acid fragment having complementarity. 6. A step of applying an aqueous liquid containing an intercalator having a fluorescent group or a conductive group and a nucleic acid fragment sample to the surface of the DNA chip according to claim 4.
A nucleic acid fragment sample complementary to the DNA fragment immobilized on the NA chip is DNized by hybridization.
Step of fixing on A chip, and DN of DNA chip
A DNA fragment on a DNA chip comprising a step of detecting fluorescence emitted from a fluorescence-generating group of an intercalator incorporated in a hybrid structure formed from the A fragment and a nucleic acid fragment sample, or a current flowing through a conductive group. A method for detecting a nucleic acid fragment having complementarity to